Voltage-gated Ca2+ channels (VGCCs) mediate Ca2+ entry into neurons and are vital for brain functions. Mutations in VGCCs cause a variety of neurological disorders, including ataxia, migraine and epilepsy. The activity of VGCCs is constantly regulated by neurotransmitters and hormones in response to various internal and external stimuli, and such regulation profoundly affects Ca2+ signaling inside cells and hence brain functions. This research proposal seeks to understand the molecular and cellular mechanisms of regulation of N- and P/Q-type of VGCCs channels by phosphatidylinositol 4,5-bisphosphate (PIP2), a membrane lipid critical for signal transduction, cytoskeletal organization and membrane trafficking. Our preliminary studies show that PIP2 exerts two distinct and opposing actions on the P/Q-type Ca2+ channels. On the one hand, PIP2 produces a strong voltage-dependent inhibition by altering the voltage-dependence of channel activation, an effect that can be prevented and reversed by PKA phosphorylation. On the other hand, PIP2 is crucial for maintaining the activity of the channels in intact cells. Furthermore, we discovered a mutation on a P/Q-type channel that dramatically reduces rundown of channel activity in excised membrane patches. We will build on these findings and carry out three areas of research. (1) We will investigate the molecular events and mechanisms underlying the dual actions of PIP2. Biochemical and site-directed mutagenesis studies will be used to identify regions and amino acids in the P/Q-type channel that are involved in PIP2 binding. (2) We will study the molecular mechanism of Ca2+ channel rundown. Specifically, we will investigate how the mutation mentioned above reduces rundown and whether its effect is linked to PIP2. (3) We will test the proposal that some well-known signaling pathways that regulate VGCCs do so by altering PIP2 interactions with the channels. Specifically, we will investigate whether G proteins inhibit Ca2+ channels by increasing the affinity of the channel for PIP2. This research may not only enhance our understanding of the complex regulatory pathways for VGCCs but also provide valuable information for the development of potential therapeutic reagents that target these pathways